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Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741

Tao Song1, Naoya Hatano, Mariko Horii

  • 1Department of Cell Physiology, Kagawa University, Faculty of Medicine, 1750-1 Ikenobe, Miki-cho, Kida-gun, Kagawa 761-0793, Japan.

FEBS Letters
|July 15, 2004
PubMed

Insights

Neuronal nitric-oxide synthase (nNOS) is inhibited by Ca2+/calmodulin-dependent protein kinase I (CaM-K I) phosphorylation at Ser741. This novel cross-talk impacts nNOS activity and calcium-calmodulin binding.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Neuroscience

Background:

  • Neuronal nitric-oxide synthase (nNOS) plays critical roles in neuronal function.
  • Regulation of nNOS activity is essential for cellular processes.
  • Calcium/calmodulin-dependent protein kinases (CaMKs) are key signaling enzymes.

Purpose of the Study:

  • To investigate the regulatory interaction between nNOS and CaM-K I.
  • To identify the specific site and mechanism of nNOS regulation by CaM-K I.
  • To explore potential cross-talk between nNOS and CaMK signaling pathways.

Main Methods:

  • Site-directed mutagenesis of nNOS (Ser741 to Ala and Asp).
  • In vitro kinase assays to assess nNOS phosphorylation by CaM-K I.
  • Measurement of nNOS activity in transfected cells.
  • Analysis of CaM binding to wild-type and mutant nNOS.

Main Results:

  • Constitutively active CaM-K I phosphorylated and inhibited nNOS at Ser741.
  • Mutation of Ser741 to Ala abolished nNOS phosphorylation and inhibition by CaM-K I.
  • Mutation mimicking phosphorylation (Ser741 to Asp) decreased CaM binding and activation.
  • Wild-type CaM-K I, but not CaM-K II or CaM-K IV, phosphorylated nNOS at Ser741.
  • nNOS activity was inhibited by 60-70% upon CaM-K I-mediated phosphorylation.

Conclusions:

  • CaM-K I directly phosphorylates and inhibits nNOS at Ser741.
  • Phosphorylation of Ser741 by CaM-K I impairs CaM binding and nNOS activation.
  • This identifies a novel regulatory mechanism and cross-talk between nNOS and CaM-K I signaling.

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